In the 1940s in the small town of Jupiter Hollow, two sets of identical twins are born in the same hospital on the same night. One set to a poor local family and the other to a rich family just passing through. The dizzy nurse on duty accidentally mixes the twins unbeknown to the parents. Our story flashes forward to the 1980s where the mismatched sets of twins are about to cross paths.

The eccentric and childish Pee-wee Herman embarks on a big adventure when his beloved bicycle is stolen. Armed with information from a fortune-teller and a relentless obsession with his prized possession, Pee-wee encounters a host of odd characters and bizarre situations as he treks across the country to recover his bike.